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The ovaries are a pair of female reproductive organs that are involved in the production of gametes “oogenesis” and the production of hormones “steroidogenesis” to mediate the oestrous cycle, preparation of the uterus for pregnancy and the preparation of the breast for lactation www.lab.anhb.uwa.edu.au/mb140/corepages/femaleRepro). The oestrous cycle is controlled by the release of the pituitary gonadotrophins, follicle stimulating hormone (FSH) and lutenising hormone (LH). The physiological responses of ovarian steroidogenesis, oogenesis, folliculargenesis, ovulation and lutenisation to the gonadotrophins are accomplished by the activation of greater than one hundred ovarian target genes (Hunzicker-Dunn et al 2006). These processes are dependant on the interplay between the steroid/gonadotrophin hormone signalling pathways and the peptide signalling pathways that cross talk and intercommunicate to induce these ovarian genes (Richards J S et al 2002) Two such critical pathways are the P13K pathway and the Ras/Raf/MEK/ERK cascade. FSH stimulates these pathways in a unique time dependant and synergistic manner (Campbell et al 1998) (Wayne et al 2007). Ras is a G protein known as Rat sarcoma homologue and a well known proliferative factor where oncogenic Ras is associated with approximately 20-30% of all human cancers (Bos 1989). Ras plays a key role in both pathways as it stimulated the Ras/Raf/MEK/ERK pathway directly and acts as a supportive activator of the P13K cascade (Wayne et al 2007). Ras works by transducing extracellular ligand mediated stimuli such as growth factors, cytokines and hormones that hit receptor tyrosine kinases (RTK’s), non receptor tyrosine kinases (NRTK’s) and G protein coupled receptors (GPCR’s) to mediate signal transduction that influences growth, differentiation and apoptosis. Ras serves as a binary molecular switch and its bioactivity is controlled by a regulated GDP/GTP cycle (Campbell SL et al 1998). IN this study the K-Ras isoform was engineered to be made constitutively active in the ovary. By introducing a glycine to aspartic acide mutation at residue 12 of the ras gene. Ras was locked in a constitutively active GTP state and thus the mutation deemed Ras to be insensitive to GTPase activating protein (GAP) stimulation. (Bourne et al 1990). A Lox-Stop-Lox K-ras conditional mouse strain capable of controlled timing and location of constitutively active Ras expression was utilised (Tuveson et al 2004) (Jackson et al 2001). The main objectives of this project were to study the effects of constitutively active Ras on ovarian cell function and morphology, to study the effects of constitutively active Ras on vital ovarian signalling genes Fshr, Lghcr and Mkp-3 and to study the effects of constitutively active Ras on p-ERK and p-PKB; protein intermediates of the Ras/Raf/MEK/ERK cascade and the P13K cascade respectively. These studies were conducted on LSL-K-ras G12D; Amhr2-Cre knock-in and LSL-K-ras G12D; Cyp19-Cre transgenic mouse models. Results showed that engineering Ras to be constitutively active causes an ovulatory malfunction. Possible reasons include the formation of tumour like follicles, follicles with odd shaped oocytes and abnormal protein and mRNA expression, reduced oocyte number, down regulation of donadotrophin receptors and or the up regulation of th Mkp-3 gene a phosphatise and newly suspected negative control component of the Ras/Raf/MEK/ERK pathway. Control of the P13K cascade is critical to maintaining the proliferative and cell survival balance. PTEN is a negative regulator involved in the control of the P13K pathway. Thus the secondary objectives of this study were to study the effect of a Pten conditional knock out on ovarian morphology and to study the effect of a Pten/K-ras double knock out on ovarian morphology using Pten flox/flox; Amhr2crel+ and Pten flox/flox; K-ras flox/+ Amhrs2 crel+ mouse models respectively. Conditional Pten know out morphology studies demonstrated a luteal cell pre tumour phenotype and the K-Ras/Pten double knock out studies presented with thecal cell thickening suggesting that both the Pten and K-ras gene products work together to augment signalling control and uphold a normal ovarian physiology.
Cahill, Nicola (Thesis), "Ovarian signalling: insights into the P13K/PKB/Foxo and the Ras/Raf/MEK/ERK pathways" (2008). Masters. Paper 49.